The long term goal of this proposal is to understand the molecular mechanisms of action of insulin. A key question is how the insulin receptor alters the activities and phosphorylation states of protein kinases to exert its ultimate physiological regulatory functions. During the past granting period, we have begun to discover the complex network of protein kinases that are regulated by the insulin receptor to cause its diverse actions. We have used and will continue to use the phosphorylation of ribosomal protein S6 as the probe for pathways, because its extensive phosphorylation is a convenient marker of insulin action. We have traced this pathway backwards two steps from S6 to an insulin-sensitive S6 protein kinase and from it to an insulin-sensitive kinase, ERK1, that phosphorylates and activates the S6 kinase. Recently, we have isolated cDNAs that encode ERK1 and a closely related kinase, ERK2. From Southern analysis and sequencing of other clones, there are may be at least two additional members of this family. To understand the control of this signal transduction pathway, we propose to define each step in the cascade that regulates the S6 kinase as follows. 1. We will identify kinases that activate ERK1. To link ERK1 to the insulin receptor we will determine whether or not the insulin receptor activates it directly or if there are additional intermediates. We have evidence indicating that a soluble kinase activates ERK1, suggesting that the soluble kinase is an intermediate in the pathway for regulation of ERK1. 2. We will identify the sites of phosphorylation on ERK1 and ERK2, because mapping the phosphorylation sites is one means of determining which protein kinase activate ERKs. This information will enable us to correlate the phosphorylation of specific sites on ERK1 and ERK2 with changes in their activities and with the kinases that phosphorylate these sites. 3. We will determine the specificity of the ERKs by testing which of other ERKs activate S6 kinase and what sites on S6 kinase are phosphorylated. 4. We will determine if one or all of the ERKs are insulin-stimulated enzymes and if they are regulated by the same or different mechanisms. These studies will provide important information about early steps in protein kinase cascades triggered by insulin.